Nobuhiro Morisaki

In Vivo Effect of TGF-β1: Enhanced Intimal Thickening by Administration of TGF-β1 in Rabbit Arteries Injured With a Balloon Catheter

Abstract The in vivo effect of transforming growth factor–β1 (TGF-β1) was studied in a model system in which arterial intimal thickening was induced by injury of rabbit arteries with a balloon cath...

Fatty acid specificity in the inhibition of cell proliferation and its relationship to lipid peroxidation and prostaglandin biosynthesis

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Cells at passage level 4 were treated with different series of fatty acids belonging to the n-9, n-6 and n-3 families. Lipid peroxidation was measured by the thiobarbituric acid assay and prostaglandin biosynthesis was measured by the radioimmunoassay of PGE and 6-keto-PGF1α. Cell proliferation was estimated from the total cell number of cultures seeded at low density. 18∶1(n-9) did not form lipid peroxides and this fatty acid stimulated cell proliferation. All fatty acids which generated lipid peroxides inhibited cell proliferation, but inhibition was correlated with the degree of lipid peroxidation only in the n-9 fatty acid family. 22∶4(n-6) and 22∶6(n-3) inhibited prostaglandin biosynthesis. 18∶2(n-6), 18∶2(n-9), 18∶3(n-3), 20∶2(n-9), 20∶3(n-3) and 20∶5(n-3) had no effect on prostaglandin biosynthesis. 18∶3(n-6), 20∶3(n-6) and 20∶4(n-6) generated prostaglandins. 20∶3(n-9) generated metabolites with prostaglandin immunoreactivity. The inhibition of cell proliferation did not correlate with enhanced or inhibited prostaglandin synthesis. The inhibition of cell proliferation was related to the structures of the different polyunsaturated fatty acid families decreasing in the order n-9>n-6>n-3. Eicosatrienoic acids were the most effective inhibitors of cell proliferation in each fatty acid family and 20∶3(n-9) was the most potent eicosatrienoic acid. These data show that specific as yet unrecognized products of fatty acid metabolism are responsible for the inhibition of cell proliferation.

Expression of LR11, a Mosaic LDL Receptor Family Member, Is Markedly Increased in Atherosclerotic Lesions

Receptors belonging to the LDL receptor (LDLR) family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. Here, we report that the expression of a 250-kDa mosaic LDLR family member, which we called LR11 for the presence of 11 ligand-binding repeats, is markedly induced during the process of atherogenesis in 2 animal models. Analysis by reverse transcription-polymerase chain reaction and RNase protection assays revealed that LR11 transcript levels rise in rabbit aortas displaying atheromatous lesions after the rabbits have been fed a high-cholesterol diet. Immunohistochemistry demonstrated that the highest induction of LR11 occurs in intimal smooth muscle cells (SMCs), followed by medial SMCs close to the intimal border of the atheromatous lesions. Experimental intimal hyperplasia by endothelial denudation showed that LR11 mRNA levels were also increased in the arteries after balloon injury, with the transcripts localized primarily in the hyperplastic intimal layer. In agreement with the correlation of LR11 induction during increased cell proliferation, cultured SMCs showed an increase in LR11 expression in the proliferative phase. Furthermore, Northern and Western blot analyses showed that medium conditioned by the monocyte-macrophage cell line THP-1 enhanced LR11 expression in cultured SMCs. These findings suggest that upregulation of LR11 might be contributing to the pathological roles of intimal and medial SMCs during arteriosclerotic lesion development and provide the first insight into the as yet unknown functional significance of this intriguing LDLR family member.

Fatty acid metabolism and cell proliferation. V. Evaluation of pathways for the generation of lipid peroxides

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Confluent cells at passage level 4–6 were challenged with arachidonic acid and treated with a number of antioxidants and inhibitors of specific lipid peroxidation pathways. Lipid peroxidation was measured by the thiobarbituric acid test for malondialdehyde (MDA) and the isolation of hydroperoxy fatty acids (HPETE) by high performance liquid chromatography (HPLC). Prostanoids were measured by radioimmunoassay and the separation of labeled compounds by HPLC. MDA, 6-keto-PGF1α, and PGE2 were formed when cells were challenged with arachidonic acid and these cells synthesized small amounts of one HPETE isomer, 15-HPETE. The HPETE isomers characteristic of the lipoxygenase pathway, 12-HPETE and 5-HPETE, were not detected. Furthermore, the lipoxygenase inhibitors, eicosatetraynoic acid (ETYA) and 6,7-dihydroxycoumarin (Esculetin), did not block MDA formation. These data show that MDA is not generated in the cells by a lipoxygenase pathway. The cyclooxygenase inhibitors, indomethacin and ETYA, did not block MDA formation but these agents blocked the formation of 15-HPETE. These data show both that 15-HPETE is generated by a cooxidation pathway and that 15-HPETE and cooxidation are not involved in MDA formation. Three inhibitors of cytochrome P450 linked lipid peroxidation, 2-amino-3-ethoxycarbonyl-6-benzyl-4, 5,6,7-tetrahydrothieno-[2,3-C]-pyridine (Tinoridine), 3-methyl-1,2-di-3-pyridyl-1-propanone (Metyrapone) and phenobarbital, did not block MDA formation. These data support earlier studies that indicated that MDA is not generated by a P450 pathway. Cells contained a bound precursor that decomposed to MDA when cells were treated with Fe3+. The cells exhibited autofluorescence and concentric lamellae in lipid droplets that are characteristic of ceroid-lipofuscin. These observations are consistent with lipid peroxidation through increased peroxisomal activity leading to the generation of MDA and the accumulation of ceroid-lipofuscin. The natural antioxidants, vitamin E and vitamin E quinone (EQ), and the synthetic antioxidants, butylated hydroxytoluene and nordihydroguaiaretic acid (NDGA), α-naphthol (α-N) and propyl gallate (PrGa), all blocked MDA formation in confluent smooth muscle cells, showing that these antioxidants did not function solely as specific inhibitors of lipoxygenase, cooxidation or P450 pathways. Cell proliferation was measured in cells challenged with arachidonic acid and treated with antioxidants and other inhibitors. The least cytotoxic and most potent antioxidant, EQ, blocked MDA formation in confluent cells and promoted growth in proliferating cells when it was present in either system in the same concentration range. The synthetic antioxidants, NDGA, α-N and PrGa, blocked prostanoid synthesis and promoted growth in proliferating cells. The cyclooxygenase inhibitors, indomethacin, ETYA and Esculetin, did not enhance cell proliferation even though they were highly effective inhibitors of prostanoid synthesis. These data suggest, but do not prove, the hypothesis that cell proliferation is controlled in part by general peroxidation reactions rather than the specific peroxidation reaction involved in prostanoid synthesis.

Fatty acid metabolism and cell proliferation. IV: Effect of prostanoid biosynthesis from endogenous fatty acid release with cyclosporin-A

Cyclosporin-A (Cyc-A) stimulates prostanoid (PGI2) synthesis in confluent smooth muscle cells from guinea pig aorta through the release of endogenous fatty acid. Cyc-A, like other stimulatory agents for prostanoids, promotes smooth muscle cell proliferation and prostanoid synthesis in these proliferating cells. Indomethacin, a cyclooxygenase inhibitor, and exogenous arachidonic acid block the Cyc-A effect on cell proliferation.

Characteristics of cyclosporine induction of increased prostaglandin levels from human peripheral blood monocytes

Human monocytes (MØ) exposed to 0.5–20 ug/ml of cyclosporine (CsA) produced levels of prostaglandins of the E series (PGE) that were 2–3-fold greater than control MØ cultured in medium alone. Maximal PGE levels were obtained at 24–48 hr incubation, and the failure to observe a linear increase of PGE levels at higher CsA concentrations appeared partially related to cytotoxic effects. CsA was considerably less effective than phorbol myristate acetate or bacterial lipopolysac-charide in increasing PGE production, but the PGE levels achieved with CsA approximated those known to suppress immune responsiveness. Other experiments showed that, although the increased PGE production with CsA was indomethacin-sensitive, CsA mostly functioned to increase the availability of free arachidonic acid (AA) instead of accelerating AA conversion by the cyclooxygenase pathway. Thus CsA can alter MØ physiology, and these alterations might inhibit quite early events during the induction phase of immune responses.

Fatty acid metabolism and cell proliferation. VII. Antioxidant effects of tocopherols and their quinones

The antioxidant capacities of α- and γ-tocopherols (α-E and γ-E) and their quinones (α-EQ and γ-EQ) were determined in non-biological and biological systems. The non-biological system consisted of arachidonic acid [20∶4 (n−6)], the oxidant cumene hydroperoxide, and a Fe3+ catalyst to facilitate malondialdehyde (MDA) formation from lipid peroxides. α-E and γ-E had similar antioxidant capacities in this system. α-EQ also functioned as an antioxidant, while γ-EQ exhibited a crossover effect by functioning as an antioxidant at low concentrations and a prooxidant at high concentrations. Biological lipid peroxidation in smooth muscle cells challenged with 20∶4 (n−6) was measured both by MDA formation in confluent cultures and by cell growth in proliferating cultures. α-E, γ-E and α-EQ had similar antioxidant capacities, but γ-EQ was highly cytotoxic for cells in both confluent and proliferating cultures. Cellular retention of antioxidants was estimated indirectly from MDA formation when cells were loaded with an antioxidant (preincubation) and then incubated for varying periods of time in fresh media containing 20∶4 (n−6). Cellular retention also was measured directly with tritiated α-E and tritiated αEQ. These studies showed that cellular retention decreased in the sequence γ-E>α-E>α-EQ. Thus, cellular retention does not explain the enhanced antioxidant capacity of α-E compared to γ-E that has been reported for animal systems. The antioxidant capacity of αE evidently is enhanced by its metabolism to a quinone which, unlike the quinone from γ-E, functions as a biological antioxidant.

Fatty acid metabolism and cell proliferation. III. Effect of prostaglandin biosynthesis either from exogenous fatty acid or endogenous fatty acid release with hydralazine

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Cells were seeded at from 40 to 80 cells per cm2 and cloned for 8 days. Media were analyzed for PGI2 (6-keto-PGF1α) using radioimmunoassay. Prostanoids were synthesized when cells were grown in media alone. Arachidonic acid stimulated prostanoid synthesis and promoted cell proliferation. Indomethacin blocked prostanoid synthesis and abolished the stimulatory effect of arachidonic acid on cell proliferation. Hydralazine stimulated fatty acid release and prostanoid synthesis in confluent cells. Hydralazine also stimulated prostanoid synthesis and promoted proliferation in growing cells. Indomethacin blocked prostanoid synthesis and abolished the stimulatory effect of hydralazine on cell proliferation.

Increased Blood Plasminogen Activator Inhibitor-1 and Intercellular Adhesion Molecule-1 as Possible Risk Factors of Atherosclerosis in Werner Syndrome

Werner syndrome is a rare premature aging syndrome accompanied by severe atherosclerosis. The etiology of atherosclerosis is suspected to be due to its complications, namely diabetes mellitus, hyperinsulinemia and hyperlipidemia. But from an autopsy case we found that some other risk factors may be involved in the mechanism of atherosclerosis in this syndrome. Previously we revealed that the plasminogen activator inhibitor-1 (PAI-1) gene was being overexpressed in skin fibroblasts from a patient with this syndrome. PAI-1 is a potent inhibitor of tissue plasminogen activator and a possible risk factor of atherosclerosis. This led us to assess the plasma concentration of PAI-1. Our working hypothesis was that the PAI-1 gene was upregulated or not fully suppressed in cells responsible for the production of PAI-1 in plasma as well as in fibroblasts. The results show a high concentration of plasma PAI-1. One of the well-known physiological substances that induce the PAI-1 gene is tumor necrosis factor-alpha, which also induces other possible risk factors of atherosclerosis, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1. We found the serum concentrations of ICAM-1 to be elevated in patients with this syndrome. We conclude that high concentrations of PAI-1 and ICAM-1 in blood may be one of the potent causes of severe atherosclerosis in Werner syndrome.

Inhibitory Effect of Ginsenosides on Migration of Arterial Smooth Muscle Cells

Migration of arterial smooth muscle cells (SMC) in the arterial wall plays an important role in the formation of intimal thickening of atherosclerotic lesions. In this study, we examined the effect of ginsenosides on SMC migration induced by platelet-derived growth factor (PDGF) and SMC-derived migration factor (SDMF). Ginsenosides had inhibitory effects on SMC migration and the striking effects were observed with ginsenoside-Rb2 and -Rc in a dose-dependent manner. These results suggest that the administration of ginsenosides on the patients may prevent intimal thickening, in part, by inhibiting SMC migration in the arterial wall.